Pharmaceutical preparation containing at least folic acid or a folate and tetrahydrobiopterin (BH4) or derivatives thereof used for the treating or preventing cardiovascular or neurological disorders by modulation of the activity of nitric oxide synthase (NOS)

ABSTRACT

The invention relates to the use of at least folic acid or a folate and tetrahydrobiopterin (BH 4 ) or derivatives thereof for treating or preventing cardiovascular or neurological disorders by modulation of the activity of nitric oxide synthase (NOS).  
     The present invention also relates to the use of at least folic acid or a folate and tetrahydrobiopterin (BH 4 ) or derivatives thereof for the production of a pharmaceutical preparation suitable for influencing the nitric oxide (NO) level, particularly by modulation of the activity of nitric oxide synthase (NOS) by reducing superoxide (O 2 ) production and enhancing nitric oxide (NO) synthesis. This effect occurs in absence of negative changes in other risk factors, e.g. lipids, blood pressure and homocysteine. Clinical areas of application include all anomalies of the nitric oxide level, particularly the prevention and treatment of cardiovascular and of neurological disorders.  
     The present invention also relates to pharmaceutical preparations comprising at least one compound selected from the group consisting of 5-formyl-(6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolic acid, 5,10-methenyl-(6R)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolic acid, 5-formimino-(6S)-tetrahydrofolic acid or (6S)-tetrahydrofolic acid or pharmaceutically compatible salts thereof, together with tetrahydrobiopterin (BH 4 ) and with pharmaceutically compatible active and adjuvant substances, such as L-arginine, for influencing the nitric oxide (NO) level.

[0001] The invention relates to the use of at least folic acid or afolate and tetrahydrobiopterin (BH₄) or derivatives thereof for treatingor preventing cardiovascular or neurological disorders by modulation ofthe activity of nitric oxide synthase (NOS). The present invention alsorelates to the use of at least folic acid or a folate andtetrahydrobiopterin (BH₄) or derivatives thereof for the production of apharmaceutical preparation suitable for influencing the nitric oxide(NO) level, particularly by modulation of the activity of nitric oxidesynthase (NOS) by reducing superoxide (O₂) production and enhancingnitric oxide (NO) synthesis. This effect occurs in the absence of anynegative changes in other risk factors, e.g. lipids, blood pressure andhomocysteine. Clinical areas of application include all anomalies of thenitric oxide level, particularly the prevention and treatment ofcardiovascular and of neurological disorders. The present invention alsorelates to pharmaceutical preparations comprising at least one compoundselected from the group consisting of 5-formyl-(6S)-tetrahydrofolicacid, 5-methyl-(6S)-tetrahydrofolic acid,5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolicacid, 5-formimino-(6S)-tetra-hydrofolic acid or (6S)-tetrahydrofolicacid, together with tetrahydrobiopterin (BH₄) or pharmaceuticallycompatible salts thereof and with pharmaceutically compatible active andadjuvant substances, such as arginine for influencing the nitric oxide(NO) level.

[0002] Within this text the term a folate or a derivative thereof, ifnot explicitly defined otherwise, always refers to the natural andunnatural stereoisomeric form of each substance, pharmaceuticallycompatible salts thereof and any mixtures of the isomers and the salts.As drugs, tetrahydrofolates have predominantly been used hitherto as thecalcium salt of 5-formyl-5,6,7,8-tetrahydrofolic acid (leucovorin) or of5-methyl-5,6,7,8-tetrahydrofolic acid (MTHF) for the treatment ofmegaloblastic folic acid deficiency anemia, as an antidote forincreasing the compatibility of folic acid antagonists, particularly ofaminopterin and methotrexate in cancer chemotherapy (“antifolaterescue”), for increasing the therapeutic effect of fluorinatedpyrimidines and for the treatment of autoimmune diseases such aspsoriasis and rheumatoid arthritis, for increasing the compatibility ofcertain antiparasitic agents, for instancetrimethoprim-sulfamethoxazole, and for decreasing the toxicity ofdideazatetra-hydrofolates in chemotherapy and for influencing thehomocysteine level, particularly for assisting the remethylation ofhomocysteine.

[0003] The term tetrahydrobiopterin (BH₄) or a derivative thereof, itnot explicitly defined otherwise, always refers to all natural andunnatural stereoisomeric forms of tetrahydrobiopterin, pharmaceuticallycompatible salts thereof and any mixtures of the isomers and the salts.The term tetrahydrobiopterin also includes any precursors oftetrahydrobiopterin, especially 7,8-dihydrobiopterin.(6R)-tetrahydrobiopterin is a naturally occuring cofactor of thearomatic amino acid hydroxylases and is involved in the synthesis of thethree common aromatic amino acids tyrosine, phenylalanine, tryptophanand the neurotransmitters dopamine and serotonin. It is also essentialfor nitric oxide synthase catalysed oxidation of L-arginine toL-citrullin and nitric oxide. Tetrahydrobiopterin is involved in manyother biochemical functions, many of which have been just recentlydiscovered.

[0004] The term arginine, if not explicitly defined otherwise, alwaysrefers to the natural and unnatural stereoisomeric form of arginine.L-arginine, a natural amino acid, is the precursor of endogenous nitricoxide (NO), which is a ubiquitous and potent vasodilator acting via theintracellular second-messenger cGMP. In healthy humans, L-arginineinduces peripheral vasodilation and inhibits platelet aggregation due toan increased NO production. Both an excess and a lack of production ofNO have been linked to pathological conditions, including cardiovasculardisorders, septic shock, inflammation and infection, and brain damage instroke and neurological disorders.

[0005] The term nitric oxide synthase (NOS), if not explicitly definedotherwise, always refers to all isoforms endothelial nitric oxidesynthase (eNOS), neuronal nitric oxide synthase (nNOS) and induciblenitric oxide synthase (iNOS).

[0006] Nitric oxide (NO) has been identified as a mediator ofatherosclerosis. Therefore it is a therapeutic target in cardiovascularprevention trials. It also plays an important role in neurologicaldisorders. Biological effects of nitric oxide (NO) are not limited tovascular relaxation, but are also important in the respiratory,urogenital and gastrointestinal system, central and peripheral nervoussystem, neuroendocrine and endocrine systems, and nonspecific immunity.

[0007] Nitric oxide (NO) and superoxide (O₂) are cytotoxins on theirown, yet it has been demonstrated that the two relatively unreactiveradicals can rapidly combine (k=3.7×10⁷ M⁻¹ s⁻¹) under physiologicalconditions to the strong oxidizing agent peroxynitrite (ONOO⁻). Thisreaction is about 3 times faster than the detoxifying catabolism ofsuperoxide by superoxide dismutase (SOD). It is believed that theformation of peroxynitrite is an important factor in the oxidativedamage associated with ischemia/reperfusion. A variety of pathologiesare associated with the formation of peroxynitrite. Peroxynitrite isinvariably formed in larger amounts when more NO is produced, and/orwhen an elevated level of superoxide prevails. In this regard,pathologies such as diabetes, atherosclerosis, and ischemia-reperfusioninjury, are associated with oxidative stress characterized by anelevated level of superoxide that can lead to increased peroxynitriteformation. Also when glutathione detoxification mechanism againstperoxynitrite is impaired critical concentrations of peroxinitrite mayoccur. Recent evidence also suggests multiple sclerosis and Alzheimer'sdisease are associated with peroxynitrite formation. In addition,peroxynitrite has also been implicated during sepsis and adultrespiratory distress syndrome. Ischemia and reperfusion are accompaniedby an increase in superoxide due to the activation of xanthine oxidaseand NAPDH oxidase, respectively. Thus, peroxynitrite is likely to beimplicated in a number of pathologies in which an imbalance of NO andsuperoxide occurs.

[0008] Several factors can contribute to reduced bioavailability of NO,ranging from impaired production to increased degradation, depending onthe risk factors involved. NO is synthesized by dimers of the 130 kDenzyme endothelial NO synthase in a reaction where arginine is oxidizedto NO and citrulline. It has been shown that eNOS produces superoxideradicals as well as NO. Under physiological conditions, NOSpredominantly produces NO, controlled by the regulatory co-enzymecalmodulin, the substrate arginine and the cofactor tetrahydrobiopterin(BH₄). Under pathophysiological conditions, such as dyslipidemia,production shifts from NO to superoxide. Clinical studies have shownimpaired NO bio-availability in patients with (risk factors for)atherosclerosis. Evidence has accumulated showing that increasedproduction of superoxide and increased degradation of NO by superoxide,rather than impaired formation of NO is the predominant cause ofimpaired NO bioavailability in early atherosclerosis. These observationsindicate that atherogenesis is linked to a pathological imbalancebetween NO and superoxide, rather than reduced NO production per se.

[0009] The level of superoxide can be lowered by substances showing arelevant scavenging capacity for superoxide radicals. Measurementsrevealed that arginine does not react with superoxide. However, botharginine and tetrahydrobiopterin (BH₄) are required to minimize orabolish superoxide formation by NOS. Tetrahydrobiopterin (BH₄) shows areaction rate with superoxide which is roughly 2 fold smaller than thatof the potent antioxidant ascorbic acid and for folic acid, folates orderivatives thereof (as an example 5-methyl-(6S)- and(6R)-tetrahydrofolic acid have been measured), the reaction rates areabout 20 times slower than that of ascorbic acid. Beside of its tenfoldlower scavenging capacity folic acid, folates or derivatives thereof aredifferent from tetrahydrobiopterin (BH₄) or derivatives thereof in thatachievable plasma concentrations are far lower. Upon standard oralsuppletion of folic acid (5 mg p.o.) systemic plasma concentrations of5-methyltetrahydrofolic acid up to ca. 150 nM are achieved whereas uponintra-arterial infusion values of 250 nM were reached. Both theseinterventions have been shown to result in an improvement inNO-availability in hypercholesterolemic patients. Still these levels offolic acid, folates or derivatives thereof remain orders of magnitudebelow those of ascorbic acid (concentrations up to 50 μM).

[0010] Despite of the situation that it has been known that “ascavenging effect of BH₄ had been remarked” [Vasquez-Vivar, J. et al.,Proc. Natl. Acad, Sci. U.S.A., 1998, 95, 9220-9225], “exogenous BH₄ iscapable of restoring impaired NO activity in prehypertensive rats”[Cosentino, F. et al., J. Clin. Invest., 1998, 101, 1530-1537],“exogenous BH₄ is capable of restoring impaired NO activity inhypercholesterolemia patients” [Stroes, E. et al., J. Clin. Invest.,1997, 99, 41-46], “exogenous BH₄ is capable of restoring impaired NOactivity in diabetic patients” [Pieper, G. M., J. Cardiovasc.Pharmacol., 1997, 29, 8-15], “folate therapy improves NO activity duringhypercholesterolemia in vivo” [Woo, K. S. et al., Circulation, 1998, 97,1-165-166] and [Verhaar, M. C. et al., Circulation, 1998; 97 (3),237-241], “folic acid and its active form 5-MTHF restore impaired NObioavailability in dyslipidemic conditions” [Wilmink, H. et al.,Arteriosclerosis Thromb Vasc Biol, 2000; 20 (1), 185-8] and [Verhaar, M.C. et al., Circulation, 1998; 97 (3), 237-241], “clinical studies haverevealed that the impairment of endogenous vasodilator function observedwith hypercholesterolemia is reversible by administration of L-arginine”[Creager, M. A. et al., Clin Invest. 1992, 90, 1248-1253] and “Folicacid supplementation improves arterial endothelial function in adultswith realtive hyperhomocysteinemia” [Woo, K. S. et al., J. Am. Collegeof Cardiology, 1999, 34 (7), 2002-2006] the use of at least folic acidor a folate and tetrahydrobiopterin (BH₄) or derivatives thereoftogether with pharmaceutically compatible active and adjuvantsubstances, such as arginine for the production of a pharmaceuticalpreparation suitable for influencing the nitric oxide (NO) level hasneither been proposed nor described hitherto.

[0011] This is probably due to the situation that it has been postulatedthat “MTHF had no direct effect on in vitro NO production by eNOS”[Verhaar, M. C. et al., Circulation, 1998; 97 (3), 237-241].

[0012] It has been found that the use of pharmaceutical preparationscontaining at least folic acid or a folate and tetrahydrobiopterin (BH₄)or derivatives thereof influences the nitric oxide (NO) level, and inparticular affects the enzymatic activity of nitric oxide synthase (NOS)by reducing superoxide production and enhancing nitric oxide (NO)synthesis. This effect occurs in absence of negative changes in otherrisk factors, e.g. lipids, blood pressure and homocysteine.

[0013] Especially surprising is this effect as in pterin-free eNOS folicacid, a folate or a derivative thereof does not affect the enzymaticactivity of nitric oxide synthase (NOS), neither with regard to NO, norto superoxide production, whereas in partially pterin-repleted eNOSfolio acid, a folate or a derivative thereof have the claimed strongeffect on the activity of the enzyme; i.e. they enhance NO productionconcomitant with a decreased production of superoxide. The beneficialvascular effect of folio acid or a folate together with at leasttetrahydrobiopterin (BH₄) or derivatives thereof cannot be attributedsolely to direct scavenging of superoxide.

[0014] Folic acid, a folate or a derivative thereof refers to folic acid(pteroylmonoglutamate), one or more of the folylpolyglutamates,compounds in which the pyrazine ring of the pterin moiety of folic acidor of the folylpolyglutamates is reduced to give dihydrofolates ortetrahydrofolates, or derivatives of all the preceding compounds inwhich the N-5 or N-10 positions carry one carbon units at various levelsof oxidation, or pharmaceutically compatible salt thereof or acombination of two or more thereof. Especially means folic acid, afolate or a derivative thereof folic acid, dihydrofolate,tetrahydrofolate, 5-methyltetrahydrofolate,5,10-methylenetetrahydrofolate, 5,10-methenyltetrahydrofolate,5,10-formiminotetrahydrofolate, 5-formyltetrahydrofolate (leucovorin),10-formyltetrahydrofolate 10-methyltetrahydrofolate, pharmaceuticallycompatible salts thereof, or a combination of two or more thereof.

[0015] Reduced folates can be converted into one another according tothe well known folate metabolism. 5-methyltetrahydrofolic acid and thepharmaceutically compatible salts thereof are preferably used, however,since 5-methyltetrahydrofolic acid is directly involved together withtetrahydrobiopterin in such functions as the biosynthesis of dopamine,norepinephrine and serotinine by the hydroxylation of phenylalanine andthe regeneration of BH₄ by the reduction of the quinonoid7,8-dihydrobiopterin to tetrahydrobiopterin. This applies in particularwhen there is an existing methylenetetrahydrofolate reductasedeficiency, wherein this deficiency implies disorders such as restrictedfunctionality or lack of activity, for example. The existence ofthermolabile methylenetetrahydrofolate reductase should be mentionedhere as the most frequent example of a methylenetetrahydrofolatereductase deficiency. Under these circumstances, especially5-methyltetrahydrofolic acid is only available in a limited amount.

[0016] Within all folates or a derivatives thereof both the natural andthe unnatural diastereoisomers, pharmaceutically compatible saltsthereof and any mixtures of the isomers and the salts, but especiallythe natural diastereoisomeric forms such as5-methyl-(6S)-tetrahydrofolic acid are applicable.

[0017] Tetrahydrobiopterin (BH₄) refers to all the natural and theunnatural forms of tetrahydrobiopterin, pharmaceutically compatiblesalts thereof and any mixtures of the isomers and the salts, butespecially the natural diastereoisomeric form(6R)-L-erythro-tetrahydrobiopterin is applicable.

[0018] Arginine refers to the both the natural and unnatural isomericform of arginine, pharmaceutically compatible salts thereof and anymixtures of the isomers and the salts, but especially the naturalisomeric form L-arginine is applicable.

[0019] For an overview of the biochemical functions of folates andtetrahydrobiopterin regarding nitric oxide and oxidative stress see FIG.7.

[0020] Pharmaceutically compatible salts should be bothpharmacologically and pharmaceutically compatible. Pharmacologically andpharmaceutically compatible salts such as these may be alkali oralkaline earth metal salts, preferably sodium, potassium, magnesium orcalcium salts.

[0021] The expression “pharmaceutical preparations” refers to enteral(e.g. oral, sublingual or rectal), parenteral or topical (e.g.transdermal) forms. Organic or inorganic substances which do not reactwith the active ingredient can be used as supports, e.g. water, oil,benzyl alcohol, polyethylene glycol, glycerol triacetate or other fattyacid glycerides, gelatine, lecithin, cyclodextrin, carbohydrates such aslactobiose or starch, magnesium stearate, talc or cellulose. Tablets,dragees, capsules powders, syrup concentrates or drops are preferred fororal application, suppositories are preferred for rectal application,and water- or oil-based solutions or lyophilisates are preferably usedfor-parenteral application.

[0022] Suspensions, emulsions or implants can also be used, and patchesor creams can be used for topical application.

[0023] Pharmaceutical preparations for parenteral application comprisesterile aqueous and non-aqueous injection solutions of thepharmaceutically-active compounds, which are preferably isotonic withthe blood of the recipient.

[0024] These preparations may comprise stabilizers, additives for thecontrolled release of the pharmaceutically-active compounds,antioxidants, such as ascorbic acid, reduced glutathione orN-acetyl-cysteine, buffers, bacteriostatic agents and adjuvantsubstances for obtaining an isotonic solution. Aqueous and non-aqueoussterile suspensions may contain suspension additives and thickeners. Thepharmaceutical preparation may exist as a single dose- or as amultiple-dose container, as sealed ampoules for example, and may bestored as a freeze-dried (lyophilized) product and prepared for use ifneed be with a sterile liquid, for example water or salt solution.Sterile powders, granules or tablets can be used in the same manner. Allthe pharmaceutical preparations may additionally contain activecompounds which act separately or synergistically. Arginine should bementioned here, which has a synergistic effect in this application. Inthis respect, arginine can be used in a dose between 1 mg and 1 g,preferably between 1 mg and 100 mg per day, for a normal dosageapplication, and can be used in a dose between 10 mg and 1 g per day fora high dosage application.

[0025] The pharmaceutical preparation contains between 0.001 mg and 1000mg of folic acid or a folate and tetrahydrobiopterin (BH₄) orderivatives thereof together with 1 mg to 10 g of arginine per dose. Inprophylaxis, preparations are used which preferably contain between 5 μgand 1000 mg of the active ingredient per dose. In therapy, preparationsare used which preferably contain between 0.1 mg and 200 mg of folicacid or a folate and tetrahydrobiopterin (BH₄) or derivatives thereoftogether with 1 mg to 1 g of arginine per dose.

[0026] The dosage depends on the form of therapy, on the form ofapplication of the pharmaceutical preparation, and on the age, weight,nutrition and condition of the patient. Treatment may be commenced witha low dosage below the optimum amount and this may be increased untilthe optimum effect is achieved. The dosages used in prophylaxis maypreferably vary for folic acid or a folate and tetrahydrobiopterin (BH₄)between 5 μg and 1000 μg per day, particularly between 50 μg and 500 μgper day. Optimum dosages in therapy vary for folic acid or a folate andtetrahydrobiopterin (BH₄) between 0.1 mg and 100 mg per day,particularly between 0.5 mg and 5 mg per day. Application may beeffected as a single administration or as a repeated dosage.

EXAMPLES TO ILLUSTRATE THE INVENTION

[0027] Chemicals

[0028] BH₄-free bovine eNOS was obtained through expression of eNOS in Ecoli. 5-methyl-(6S)-tetrahydrofolic acid, and its stereoisomer,5-methyl-(6R)-tetrahydrofolic acid were used in purities >99.8%. Thespin trap, 5-diethoxy-phosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO)and all other used chemicals are commercially available.

[0029] Electron Paramagnetic Resonance Measurements (EPR)

[0030] The EPR spectra were recorded at 37° C. on a modified Bruker ESP300. Spin trap experiments were performed with both HX/XO and eNOS. ForHX/XO the solution contained 0.5 mM hypoxanthine, 12.5 mU/ml xanthineoxidase and 50 mM DEPMPO in phosphate buffer (pH 7.4). The eNOS assaycontained 250 nM eNOS dimers (0.065 mg protein/ml), 0.5 mM NADPH, 10 μML-arginine, 1 mM CaCl₂, 300 U/ml calmodulin and 50 mM DEPMPO inphosphate buffer (pH 7.4).

[0031] Determination of NO-Production by eNOS

[0032] NOS activity was determined by quantifying the conversion ofL-[2,3,4.5-³H]arginine into L-[2,3,4,5-³H]-citrulline. Briefly, 2 μgeNOS (BH₄-free or -repleted) was incubated during 5 min at 37° C. in 100μl HEPES buffer (pH 7.4) containing DTPA (0.1 mmol/l), CaCl₂ (0.2mmol/l), calmodulin (20 μg/ml), NADPH (0.5 mmol/l), FMN (1 μmol/l), FAD(1 μmol/l), glutathione (100 mmol/l), BSA (200 μg/ml), L-arginine (100μmol/l), and L-[2,3,4,5-³H]-arginine (3.7 KBq). All measurements wereperformed in triplicate. After correction for nonspecific activity, eNOSactivity was calculated from the percent conversion of [³H]-arginineinto [³H]-citrulline and expressed as nmoles per mg protein per min.

[0033] Cell Cultures

[0034] Microvascular endothelial bEND3 cells were cultured to confluencein 6-well culture plates for determination of nitrite or in 15-cm dishesfor electron spin resonance experiments. After the cells reachedconfluence the medium was changed to M-199 (Sigma chem), supplementedwith 0.1% BSA, 5 mM L-glutamine, antibiotics and 5-methyltetrahydrofolicacid (0, 1, 10 μM) or sepiapterin (100 μM), respectively, for 24 hours.

[0035] Determination of NO Production by Endothelial Cells

[0036] The NO production by endothelial cells was assessed byquantification of the nitrite content in the supernatant with acommercially available fluorimetric kit (Cayman Chemicals).Acetylcholine-induced NO production is presented as the differencebetween stimulated minus the unstimulated nitrite content.

[0037] Statistical Analysis

[0038] Changes in NO-production were tested with an unpaired t-test.Changes in radical adduct formation by tetrahydrobiopterin (FIG. 3) or5-methyl-(6S)-tetrahydrofolic acid (FIG. 4) were tested with analysis ofvariance. If variance ratios reached statistical significance,differences between the means were analyzed with theStudent-Newman-Keuls test for p<0.05.

[0039] 5-Methyltetrahydrofolic Acid Direct Superoxide Scavenging

[0040] XO Activity

[0041] Assessment of urate levels (assessed with a uricase-hydrogenperoxide assay) is a standard method to determine enzymatic activity ofXO. However, reductive substance, like vitamin C or NUHF, are known tointerfere with the urate determination. Basal urate levels after 60minutes of incubation (1, 2. 5 mU/ml XO, 0.5 mM hypoxanthine inphosphate buffer, pH 7.4, 37° C.) were 128±20 μM. Addition of 50 μM5-methyl-(6S)-tetrahydrofolic acid at the beginning of the incubationperiod resulted in a significantly lower urate level of 48.7±1.7 μM.Addition of 50 μM 5-methyl-(6S)-tetrahydrofolic acid at the end of theincubation period (just prior to urate assessment) resulted in a similarurate level (49.9±1.9 μM). These date show that5-methyl-(6S)-tetrahydrofolic acid interferes with the quantification ofurate, rather than the urate production itself and that5-methyltetrahydrofolic acid does not affect the rate of urateproduction by XO.

[0042] Determination of Superoxide Trapping Rates by CompetitiveSuperoxide Trapping

[0043] The trapping rates for superoxide were determined at 37° C. bycomparing the trapping efficiency of L, arginine, tetrahydrobiopterinand 5-methyl-(6S)- and -(6R)-tetrahydrofolic acid with the knowntrapping efficiency of the spin trap DEPMPO (competitive superoxidetrapping [CST]). Using HX/XO as superoxide generating system, thepresence of other compound (like L-arginine, tetrahydrobiopterin,5-methyltetrahydrofolic acid or ascorbic acid) will result in lessgeneration of DEPMPO spin adducts. Reaction channels other than withDEPMPO, L-arginine, tetrahydrobiopterin, 5-methyltetrahydrofolic acid orascorbic acid can be neglected as the adduct yield does not increasefurther if DEPMPO concentrations higher than 50 mM are used. The timecurves of the EPR intensity in the HX/XO system were described by singleexponentials with a time constant, t=10±0.5 min (FIG. 1). Plots of thesteady state limits as a function of 5-methyl-(6S)- and-(6R)-tetrahydrofolic acid concentration in the HX/XO system are givenin FIG. 2 (solid circles). Both isomers show the same linearconcentration dependence.

[0044] The reaction rates with superoxide are given by k for the testedsubstance and k_(d) for the reference compound DEPMPO respectively. Areference value of k_(d)=80 (Ms)⁻¹ has been shown to be reliable. Basedthereon the following values have been determined at pH 7.4 and 37° C.k_(ascorbic acid)/k_(d) = 4400 k_(ascorbic acid) = 3.5 × 10⁵ (Ms)⁻¹[literature value k_(ascorbic acid) = 2.7 × 10⁵ (Ms)⁻¹] k_(BH4)/k_(d) =1200 k_(BH4) = 1.5 × 10⁶ (Ms)⁻¹ k_(L-MTHF)/k_(d) =  150 k_(L-MTHF) = 1.2× 10⁴ (Ms)⁻¹ k_(D-MTHF)/k_(d) =  150 k_(D-MTHF) = 1.2 × 10⁴ (Ms)⁻¹k_(arginine)/k_(d) = <10⁻² k_(arginine) = neglibile

[0045] The presence of L-arginine did not affect the formation of DEPMPOadducts, even at high concentrations (up to 100 mM). Therefore,L-arginine has no significant scavenging capacity for superoxide.

[0046] BH₄ is an about 2 times less potent scavenger than ascorbic acid,whereas both isomers of 5-methyltetrahydrofolic acid are in this regardabout 20 times less potent than ascorbic acid. In addition the usualplasma concentration for tetrahydrobiopterin and folates are in the lownanomolar range. Upon oral supplementation, the level of folic acid maybe raised to micromolar range, which is still below the vitamin C levelsof 30-50 micromolar observed in vivo. Therefore due to the lowscavenging potency and low plasma concentration of BH₄ and folates theirdirect superoxide scavenging capacity is not relevant in vivo, whereantioxidant mechanisms like vitamin C or superoxide dismutase (SOD) havefar higher capacity for removal of superoxide. Instead, folates exertstheir beneficial effects together with tetrahydrobiopterin throughmodulation of the enzymatic activity of NOS.

[0047] 5-Methyltetrahydrofolic Acid-Pterin-Free eNOS

[0048] Superoxide Production by Pterin-Free eNOS

[0049] To elucidate whether that 5-methyl-(6S)-tetrahydrofolic acidimproves NO bioavailability in vivo in hypercholesterolemic patients bya direct effect of 5-methyl-(6S)-tetrahydrofolic acid on eNOS,competitive superoxide trapping (CST) experiments using eNOS as asuperoxide generating system have been carried aout. The effect of5-methyl-(6S)- and -(6R)-tetrahydrofolic acid on the pterin-free eNOS(FIG. 3, solid circles) coincides with the data from the HX/XOexperiments (c.f. FIG. 2). It demonstrates that for pterin-free eNOSimpaired formation of spin adducts can be fully accounted for by thecapacity of 5-methyl-(6S)- and -(6R)-tetrahydrofolic acid to scavengesuperoxide in a bimolecular scavenging reaction. In particular, thepresence of 5-methyl-(6S)- or -(6R)-tetrahydrofolic acid does not affectthe rate of superoxide production by pterin-free eNOS.

[0050] In pterin-free eNOS, folic acid, a folate or a derivative thereofsignificantly reduce the formation of DEPMPO superoxide adducts. Thedegree of reduction in superoxide activity by folic acid, a folate or aderivative thereof is equivalent to that observed in the HX/XO system.It shows that for pterin-free eNOS folic acid a folate or a derivativethereof exerts its effects through pure scavenging only, withoutinterfering with enzymatic activity. Combining the results for NO andsuperoxide, pterin-free eNOS is seen to be completely oblivious to thepresence of folic acid, a folate or a derivative thereof.

[0051] 5-methyltetrahydrofolic acid-Pterin-Repleted eNOS

[0052] Superoxide Production by eNOS.—Pterin-Repleted Case

[0053] As far as pterin-repleted eNOS is concerned (FIG. 3, opencircles), addition of 5-methyl-(6S)- or -(6R)-tetrahydrofolic acidresults in a very strong reduction in the rate of DEPMPO adductformation (as manifests itself from a much steeper slope). Thisreduction by far exceeds the reduction observed in pterin-free eNOS.This observation cannot be fully explained by the capacity of5-methyl-(6S)- and -(6R)-tetrahydrofolic acid to scavenge superoxide.Therefore the presence of 5-methyltetrahydrofolic acid reduces thesuperoxide production by eNOS in a concentration dependent way with bothstereoisomers of 5-methyltetrahydrofolic acid having the same potency.To evaluate whether 5-methyltetrahydrofolic acid only affects eNOS afterpreincubation with BH₄, increasing amounts of BH₄ have been added topterin-free eNOS in presence and absence of5-methyl-(6S)-tetrahydrofolic acid 25 μM (FIG. 4) As expected, in theabsence of 5-methyltetrahydrofolic acid, addition of BH₄ caused adose-dependent decrease in superoxide production. In pterin-free eNOS,addition of 25 μM 5-methyl-(6S)-tetrahydrofolic acid does not causesignificant changes in radical adduct formation (FIG. 4). In contrast,addition of 5-methyltetrahydrofolic acid to partially repleted eNOS(still BH₄ deficient) causes a substantial reduction in the amount ofsuperoxide adducts (FIG. 4).

[0054] NO-Production by eNOS

[0055] The NO production by pterin-free eNOS is located at the detectionlimit of the arginine-citrulline conversion assay (FIG. 5). Addition of5-methyl-(6S)- or -(6R)-tetrahydrofolic acid (100 μM final) topterin-free eNOS has no significant effect on NO production (FIG. 5). Incontrast, in pterin-repleted eNOS, two significant differences arise:first, a clear basal NO-production is observed (FIG. 5). Second, theaddition of both 5-methyl-(6S)- or -(6R)-tetrahydrofolic acid (100 μMfinal) causes a significant increase in NO production (FIG. 5; p<0.05vs. BH₄ alone).

[0056] NO-Production by Endothelial Cells

[0057] Preincubiation with 5-methyltetrahydrofolic acid did not affectnitrite release in unstimulated endothelial cells. Acetylcholinestimulation caused a significant increase in nitrite release (FIG. 6).Preincubation of endothelial cells with 5-methyl-(6S)-tetrahydrofolicacid and sepiapterin resulted in a significant-further increase inacetylcholine-induced nitrite production (FIG. 6).

[0058] The pterin-repleted eNOS used in our studies shows substantialbasal production of both NO as well as superoxide and therefore shouldbe considered as BH₄ deficient, i.e. partially uncoupled. Under theseconditions, addition of folic acid, a folate or a derivative thereofincreases NO production. Both diastereoisomeric forms of the folates andderivatives thereof have the same effect. At the same time, theformation of DEPMPO superoxide adducts is strongly reduced by both folicacid, a folate or a derivative thereof. The reduction of adductformation caused by folic acid, a folate or a derivative thereof by farexceeds that observed in the HX/XO system. This shows that the majorimpact of folic acid, a folate or a derivative thereof must be a directinterference with the enzymatic superoxide production by thepterin-repleted eNOS. Again, both diastereoisomeric forms of the folateshave comparable effects. Combining the results, for NO and superoxide,the enzymatic activity of pterin-repleted eNOS is highly sensitive tothe presence of folic acid, a folate or a derivative thereof. Theoverall effect is a substantial shift from superoxide production towardsNO production. From FIG. 2, we estimate that superoxide production byeNOS is reduced by a factor of 2 at a concentration of[5-methyltetrahydrofolic acid]=50 μM, i.e. ca.2005-methyltetrahydrofolic acid molecules per eNOS dimer. Similarmolecular ratios of 5-methyltetrahydrofolic acid vs. eNOS can beachieved in vivo upon oral supplemeatation with folic acid, a folate ora derivative thereof.

[0059] 5-methyltetrahydrofolic Acid-Pterin Interaction

[0060] It has been shown that folic acid, a folate or a derivativethereof, requires BH₄ before it can affect the enzymatic activity ofeNOS. Folic acid, a folate or a derivative thereof supports the actionas a cofactor of BH₄.

[0061] Moreover, therapy with folic acid, a folate or a derivativethereof did not show any effect on biopterin levels in vivo. Thereforefolic acid, a folate or a derivative thereof exerts its effect viaenhanced binding of BH₄ to eNOS.

[0062] Folic acid, a folate or a derivative thereof act as facilitatorof the oxidation of BH₄ to the BH₄-radical.

[0063] 5-methyltetrahydrofolic Acid-Endothelial Cells

[0064] It has been shown that the effects of folic acid, a folate or aderivative thereof on endogenous, eNOS in endothelial cells arecompatible with the findings on the recombinant enzyme. In particular,it has been shown an enhanced T40 status in cultured endothelial cellsupon 5-methyltetrahydrofolic acid suppletion.

[0065] The decreased superoxide production and enhanced NO synthesis bythe nitric oxide synthase (NOS) following the application of folic acid,a folate or a derivative thereof provides a plausible explanation forthe increased NO bio-availability in humans upon 5-methyltetrahydrofolicacid suppletion during dyslipidaemia.

Example 1

[0066] A Tablet Containing 50 mg 5-formyl-(6S)-tetrahydrofolic Acid and50 mg (6R)-tetrahydrobiopterin (BH₄)

[0067] A mixture of 665 g of the pentahydrate of the calcium salt of5-formyl-(6S)-tetrahydrofolic acid (corresponding to 500 g5-formyl-(6S)-tetrahydrofolic acid), 645 g (6R)-tetrahydrobiopterindihydrochloride (corresponding to 500 g (6R)-tetrahydrobiopterin), 4 kglactose, 1.2 kg starch, 0.2 kg talc and 0.1 kg magnesium stearate ispressed to form tablets, so that each tablet contains 50 mg5-formyl-(6S)-tetrahydrofolic acid and 50 mg (6R)-tetrahydrobiopterin(BH₄).

[0068] The tablet can be coated as a film tablet or can be ground andused in capsule form.

Example 2

[0069] A Suppository Containing 500 mg 5-methyl-(6S)-tetrahydrofolicAcid and 500 mg (6R)-tetrahydrobiopterin (BH₄)

[0070] A mixture of 632 g of the pentahydrate of the calcium salt of5-methyl-(6S)-tetrahydrofolic acid (corresponding to 500 g5-methyl-(6S)-tetrahydrofolic acid), 645 g (6R)-tetrahydrobiopterindihydrochloride (corresponding to 500 g (6R)-tetrahydrobiopterin), 50 ghydroxy-propylcellulose and 2 kg of semisynthetic glycerides is meltedto form suppositories, so that each suppository contains 500 mg5-methyl-(6S)-tetrahydrofolic acid and 500 mg (6R)-tetrahydrobiopterin(BH₄).

Example 3

[0071] An Injection Solution Containing 5 mg5-methyl-(6S)-tetrahydrofolic Acid, 1 mg (6R)-tetrahydrobiopterin (BH₄)and 5 mg L-arginine

[0072] 5.0 g 5-methyl-(6S)-tetrahydrofolic acid, 1.0 g(6R)-tetrahydrobiopterin (BH₄), 5.0 g L-arginine, 10 g glutathione, 30 gcitric acid, 160 g mannitol, 1 g methyl-p-hydroxybenzoic acid, 17.7 gsodium hydroxide (or the requisite amount in order to obtain a pH of thesolution of 7.3 to 7.8) is dissolved in 3 liters of water for injectionand introduced into ampoules, so that each ampoule contains 5 mg5-methyl-(6S)-tetrahydrofolic acid, 1 mg (6R)-tetrahydrobiopterin (BH₄)and 5 mg L-arginine.

Example 4

[0073] An Injectable Lyophilisate Containing 1 mg Tetrahydrofolic Acidand 1 mg Tetrahydrobiopterin (BH₄)

[0074] A solution of 1.05 g of the sodium salt of tetrahydrofolic acid(corresponding to 1.0 g tetrahydrofolic acid) and 1.40 g(6R)-tetrahydrobiopterin sulfate (corresponding to 1.0 g(6R)-tetrahydrobiopterin) in 1000 ml double-distilled water isintroduced via sterile filtration into ampoules and lyophilised, so thateach ampoule contains 1 mg tetrahydrofolic acid and 1 mgtetrahydrobiopterin (BH₄).

[0075] Tetrahydrofolic acid is very sensitive to oxygen, and stringentlyoxygen-free conditions therefore have to be employed. The use of anantioxidant such as ascorbic acid may be necessary.

Example 5

[0076] An Injectable Lyophilisate Containing 20 mg5,10-methylene-(6R)-tetrahydrofolic Acid and 50 mg(6R)-tetrahydrobiopterin (BH₄)

[0077] A solution of of the beta-hydroxypropyl-cyclodextrin inclusioncompound of the sodium salt of 5,10-methylene-(6R)-tetrahydrofolic acidcontaining 10 g 5,10-methylene-(6R)-tetrahydrofolic acid and 50 g(6R)-tetrahydrobiopterin (BH₄) in 2000 ml of double-distilled water isintroduced via sterile filtration into ampoules, so that each ampoulecontains 20 mg 5,10-methylene-(6R)-tetrahydrofolic acid and 50 mg(6R)-tetrahydrobiopterin (BH₄).

[0078] The same precautionary measures apply to5,10-methylene-tetrahydrofolic acid as for tetrahydrofolic acid(preceding Example).

Example 6

[0079] A Tablet Containing 4 mg 5-formyl-(6S)-tetrahydrofolic Acid and10 mg (6R)-tetrahydrobiopterin (BH₄)

[0080] A mixture of 53.2 g of the pentahydrate of the calcium salt of5-formyl-(6S)-tetrahydrofolic acid (corresponding to 40 g5-formyl-(6S)-tetrahydrofolic acid), 100 g (6R)-tetrahydrobiopterin(BH₄), 4 kg lactose, 1.2 kg starch, 0.2 kg talc and 0.1 kg magnesiumstearate is pressed to form tablets, so that each table contains 4 mg5-formyl-(6S)-tetrahydrofolic acid and 10 mg (6R)-tetrahydrobiopterin(BH₄).

[0081] The tablet can be coated as a film tablet or can be ground andused in capsule form.

Example 7

[0082] An Injectable Lyophilisate Containing 10 μg6-methyl-(6S)-tetrahydrofolic Acid and 10 μg (6R)-tetrahydrobiopterin(BH₄)

[0083] A solution of 10 mg of the sodium salt of5-methyl-(6S)-tetrahydrofolic acid and 10 mg (6R)-tetrahydrobiopterin(BH₄) in 1000 ml of double-distilled water is introduced, via sterilefiltration under an inert gas, into ampoules and lyophilised, so thateach ampoule contains 10 μg 5-methyl-(6S)-tetrahydrofolic acid and 10 μg(6R)-tetrahydrobiopterin (BH₄).

[0084] Tetrahydrofolic acid is very sensitive to oxygen, and stringentlyoxygen-free conditions therefore have to be employed. The use of anantioxidant such as ascorbic acid may be necessary.

Example 8

[0085] A Tablet Containing 15 mg 5-methyl-(6S)-tetrahydrofolic Acid and5 mg (6R)-tetrahydrobiopterin (BH₄)

[0086] A mixture of 19.18 g of the pentahydrate of the calcium salt of5-methyl-(6S)-tetrahydrofolic acid (corresponding to 15 g5-methyl-(6S)-tetrahydrofolic acid), 5 g (6R)-tetrahydrobiopterin (BH₄),120 g lactose, 21.5 g maize starch, 7.08 g acetylcellulose, 2.28 gdiethyl phthalate, 0.64 g silicone HK-15 and 2 g magnesium stearate ispressed to form tablets, so that each tablet contains 15 mg5-methyl-(6S)-tetrahydrofolic acid and 5 mg (6R)-tetrahydrobiopterin(BH₄).

[0087] The tablet can be coated as a film tablet or can be ground andused in capsule form.

Example 9

[0088] Tablets Containing 10 mg 5-methyl-(6S)-tetrahydrofolic Acid and10 mg (6R)-tetrahydrobiopterin (BH₄)

[0089] In an analogous manner to that described in Example 8, tabletscontaining 10 mg 5-methyl-(6S)-tetrahydrofolic acid and 10 mg(6R)-tetrahydrobiopterin (BH₄) are produced using maize starch, lactose,magnesium stearate, polyethylene glycol 6000, polymethacrylate,polysorbitol 80, dimethylpolysiloxane, sodium hydroxide and talc.

Example 10

[0090] A Combination Preparation Comprising 5-methyltetrahydrofolicAcid, Tetrahydrobiopterin (BH₄) and Arginine

[0091] A film tablet which contains the following constituents isformulated for preparations for oral application:

[0092] 25 mg 5-methyltetrahydrofolic acid

[0093] 25 mg tetrahydrobiopterin (BH₄)

[0094] 250 mg arginine

[0095] pharmaceutically compatible adjuvant substances

[0096] The tablet can be coated as a film tablet or can be ground andused in capsule form.

[0097] This combination preparation may also be formulated as asolution, e.g. for parenteral application.

[0098] The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples. Also, the preceding specific embodiments are to be construedas merely illustrative, and not limitative of the remainder of thedisclosure in any way whatsoever.

[0099] The entire disclosure of all applications, patents andpublications, cited above and below, are hereby incorporated byreference.

[0100] From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A method for the modulation of the activity ofnitric oxide synthase (NOS), comprising administering at least (1) folicacid or a folate; and (2) tetrahydrobiopterin (BH₄) or derivativesthereof
 2. The method as defined in claim 1 wherein folic acid or afolate or a derivative thereof is folic acid (pteroylmonoglutamate), oneor more of the folylpolyglutamates, compounds in which the pyrazine ringof the pterin moiety of folic acid or of the folylpolyglutamates isreduced to give dihydrofolates or tetrahydrofolates, or derivatives ofall the preceding compounds in which the N-5 or N-10 positions carry onecarbon units at various levels of oxidation, or salt thereof or acombination of two or more thereof.
 3. The method as defined in claim 2wherein folate or a derivative thereof is dihydrofolic acid,tetrahydrofolic acid, 5-methyltetrahydrofolic acid,5,10-methylenetetrahydrofolic acid, 5,10-methenyltetrahydrofolic acid,5,10-formiminotetrahydrofolic acid, 5-formyltetrahydrofolic acid(leucovorin), 10-formyltetrahydrofolic acid, 10-methyltetrahydrofolicacid, salts thereof, or a combination of two or more thereof.
 4. Themethod as defined in claim 3 wherein folate or a derivative thereof isin the natural diastersoisomer of foliate or a derivative thereof.
 5. Amethod of preventing or treating diseases associated with disturbedactivity of nitric oxide synthase (NOS) in the human body comprisingadministering a therapeutically effective amount of a drug containing atleast (1) folic acid or a folate; and (2) tetrahydrobiopterin (BH₄) orderivatives thereof to a human subject.
 6. The method as defined inclaim 5 wherein folic acid or a folate or a derivative thereof is folicacid (pteroylmonoglutamate), one or more of the folylpolyglutamates,compounds in which the pyrazine ring of the pterin moiety of folic acidor of the folylpolyglutamates is reduced to give dihydrofolates ortetrahydrofolates, or derivatives of all the preceding compounds inwhich the N-5 or N-10 positions carry one carbon units at various levelsof oxidation, or pharmaceutically compatible salt thereof, or acombination of two or more thereof.
 7. The method as defined in claim 6wherein folate or a derivative thereof is dihydrofolic acid,tetrahydrofolic acid, 5-methyltetrahydrofolic acid,5,10-methylenetetrahydrofolic acid, 5,10-methenyltetrahydrofolic acid,5,10-formiminotetrahydrofolic acid, 5-formyltetrahydrofolic acid(teucovorin), 10-formyltetrahydrofolic acid, 10-methyltetrahydrofolicacid, pharmaceutically compatible salts thereof, or a combination of twoor more thereof.
 8. The method as defined in claim 7 wherein at leastone of folate or tetrahydrobiopterin or a derivative thereof is in thenatural stereoisomeric form.
 9. A method for the treatment or preventionof at least moderately reduced levels of nitric oxide (NO), comprisingadministering a therapeutically effective amount of a drug containing atleast (1) folic acid-or a folate; and (2) tetrahydrobiopterin (BH₄) orderivatives thereof
 10. The method as defined in claim 9 wherein folicacid or a folate or a derivative thereof is folic acid(pteroylmonoglutamate), one or more of the folylpolyglutamates,compounds in which the pyrazine ring of the pterin moiety of folic acidor of the folylpolyglutamates is reduced to give dihydrofolates ortetrahydrofolates, or derivatives of all the preceding compounds inwhich the N-5 or N-10 positions carry one carbon units at various levelsof oxidation, or pharmaceutically compatible salt thereof, or acombination of two or more thereof.
 11. The method as defined in claim10 wherein folate or a derivative thereof is dihydrofolic acid,tetrahydrofolic acid, 5-methyltetrahydrofolic acid,5,10-methylenetetrahydrofolic acid, 5,10-methenyltetrahydrofolic acid,5,10-formiminotetrahydrofolic acid, 5-formyltetrahydrofolic acid(leucovorin), 10-formyltetrahydrofolic acid, 10-methyltetrahydrofolicacid, pharmaceutically compatible salts thereof, or a combination of twoor more thereof.
 12. The method as defined in claim 11 wherein at leastone of folate or tetrahydrobiopterin or a derivative thereof is in thenatural stereoisomeric form.
 13. A method for the treatment orprevention of at least moderately elevated levels of superoxide,comprising administering a therapeutically effective amount of a drugcontaining at least (1) folic acid or a folate; and (2)tetrahydrobiopterin (BH₄) or derivatives thereof
 14. The method asdefined in claim 13 wherein folic acid or a folate or a derivativethereof is folic acid (pteroylmonoglutamate), one or more of thefolylpolyglutamates, compounds in which the pyrazine ring of the pterinmoiety of folic acid or of the folylpolyglutamates is reduced to givedihydrofolates or tetrahydrofolates, or derivatives of all the precedingcompounds in which the N-5 or N-10 positions carry one carbon units atvarious levels of oxidation, or pharmaceutically compatible salt thereofor a combination of two or more thereof.
 15. The method as defined inclaim 14 wherein folate or a derivative thereof is dihydrofolic acid,tetrahydrofolic acid, 5-methyltetrahydrofolic acid,5,10-methylenetetrahydrofolic acid, 5,10-methenyltetrahydrofolic acid,5,10-formiminotetrahydrofolic acid, 5-formyltetrahydrofolic acid(leucovorin), 10-formyltetrahydrofolic acid, 10-methyltetrahydrofolicacid, pharmaceutically compatible salts thereof, or a combination of twoor more thereof.
 16. The method as defined in claim 15 wherein at leastone of folate or tetrahydrobiopterin or a derivative thereof is in thenatural stereoisomeric form.
 17. A method of preventing or treatingdisease associated with decreased nitric oxide levels in the human bodycomprising administering a therapeutically effective amount of a drugcontaining at least (1) folic acid or a folate; and (2)tetrahydrobiopterin (BH₄) or derivatives thereof to a human subject. 18.The method as defined in claim 17 wherein folic acid or a folate or aderivative thereof is folic acid (pteroylmonoglutamate), one or more ofthe folylpolyglutamates, compounds in which the pyrazine ring of thepterin moiety of folic acid or of the folylpolyglutamates is reduced togive dihydrofolates or tetrahydrofolates, or derivatives of all thepreceding compounds in which the N-5 or N-10 positions carry one carbonunits at various levels of oxidation, or pharmaceutically compatiblesalt thereof, or a combination of two or more thereof.
 19. The method asdefined in claim 18 wherein folate or a derivative thereof isdihydrofolic acid, tetrahydrofolic acid, 5-methyltetrahydrofolic acid,5,10-methylenetetrahydrofolic acid, 5,10-methenyltetrahydrofolic acid,5,10-formiminotetrahydrofolic acid, 5-formyltetrahydrofolic acid(leucovorin), 10-formyltetrahydrofolic acid, 10-methyltetrahydrofolicacid, pharmaceutically compatible salts thereof, or a combination of twoor more thereof.
 20. The method as defined in claim 19 wherein at leastone of folate or tetrahydrobiopterin or a derivative thereof is in thenatural stereoisomeric form.
 21. A method of preventing or treatingdisease associated with elevated superoxide levels in the human bodycomprising administering a therapeutically effective amount of a drugcontaining at least (1) folic acid or a folate; and (2)tetrahydrobiopterin (BH₄) or derivatives thereof to a human subject. 22.The method as defined in claim 21 wherein folic acid or a folate or aderivative thereof is folic acid (pteroylmonoglutamate), one or more ofthe folylpolyglutamates, compounds in which the pyrazine ring of thepterin moiety of folic acid or of the folylpolyglutamates is reduced togive dihydrofolates or tetrahydrofolates, or derivatives of all thepreceding compounds in which the N-5 or N-10 positions carry one carbonunits at various levels of oxidation, or pharmaceutically compatiblesalt thereof, or a combination of two or more thereof.
 23. The method asdefined in claim 22 wherein folate or a derivative thereof isdihydrofolic acid, tetrahydrofolic acid, 5-methyltetrahydrofolic acid,5,10-methylenetetrahydrofolic acid, 5,10-methenyltetrahydrofolic acid,5,10-formiminotetrahydrofolic acid, 5-formyltetrahydrofolic acid(leucovorin), 10-formyltetrahydrofolic acid, 10-methyltetrahydrofolicacid, pharmaceutically compatible salts thereof, or a combination of twoor more thereof.
 24. The method as defined in claim 23 wherein at leastone of folate or tetrahydrobiopterin or a derivative thereof is in thenatural stereoisomeric form.
 25. A method according to claims 1, 5, 9,13, 17 or 21 where pathophysiological conditions are present.
 26. Amethod according to claim 25, wherein the disease is a cardiovasculardisease
 27. The method as defined in claim 26 wherein folic acid or afolate or a derivative thereof is folic acid (pteroylmonoglutamate), oneor more of the folylpolyglutamates, compounds in which the pyrazine ringof the pterin moiety of folic acid or of the folylpolyglutamates isreduced to give dihydrofolates or tetrahydrofolates, or derivatives ofall the preceding compounds in which the N-5 or N-10 positions carry onecarbon units at various levels of oxidation, or pharmaceuticallycompatible salt thereof, or a combination of two or more thereof. 28.The method as defined in claim 27 wherein folate or a derivative thereofis dihydrofolic acid, tetrahydrofolic acid, 5-methyltetrahydrofolicacid, 5,10-methylenetetrahydrofolic acid, 5,10-methenyltetrahydrofolicacid, 5,10-formiminotetrahydrofolic acid, 5-formyltetrahydrofolic acid(leucovorin), 10-formyltetrahydrofolic acid, 10-methyltetrahydrofolicacid, pharmaceutically compatible salts thereof, or a combination of twoor more thereof.
 29. The method as defined in claim 28 wherein at leastone of folate or tetrahydrobiopterin or a derivative thereof is in thenatural stereoisomeric form.
 30. A method according to claim 25 whereinthe disease is a neurological disorder.
 31. The method as defined inclaim 30 wherein folic acid, a folate or a derivative thereof is folicacid (pteroylmonoglutamate), one or more of the folylpolyglutamates,compounds in which the pyrazine ring of the pterin moiety of folic acidor of the folylpolyglutamates is reduced to give dihydrofolates ortetrahydrofolates, or derivatives of all the preceding compounds inwhich the N-5 or N-10 positions carry one carbon units at various levelsof oxidation, or pharmaceutically compatible salt thereof, or acombination of two or more thereof.
 32. The method as defined in claim31 wherein folate or a derivative thereof is dihydrofolic acid,tetrahydrofolic acid, 5-methyltetrahydrofolic acid,5,10-methylenetetrahydrofolic acid, 5,10-methenyltetrahydrofolic acid,5,10-formiminotetrahydrofolic acid, 5-formyltetrahydrofolic acid(leucovorin), 10-formyltetrahydrofolic acid, 10-methyltetrahydrofolicacid, pharmaceutically compatible salts thereof, or a combination of twoor more thereof.
 39. The method as defined in claim 32 wherein at leastone of folate or tetrahydrobiopterin or a derivative thereof is in thenatural stereoisomeric form.
 34. A method according to claims 1, 5, 9,13, 17, 21, 25, 26 or 30 wherein folic acid or a folate andtetrahydrobiopterin (BH₄) or derivatives thereof is administered incombination with at least one active substance or at least one adjuvantsubstance.
 35. A method according to claim 34, wherein the activesubstance is a pharmaceutically compatible active substance.
 36. Amethod according to claim 35, wherein the pharmaceutically compatibleactive substance comprises at least arginine.
 37. The method as definedin claim 36 wherein folic acid or a folate or a derivative thereof isfolic acid (pteroylmonoglutamate), one or more of thefolylpolyglutamates, compounds in which the pyrazine ring of the pterinmoiety of folic acid or of the folylpolyglutamates is reduced to givedihydrofolates or tetrahydrofolates, or derivatives of all the precedingcompounds in which the N-5 or N-10 positions carry one carbon units atvarious levels of oxidation, or pharmaceutically compatible salt thereofor a combination of two or more thereof.
 38. The method as defined inclaim 37 wherein folate or a derivative thereof is dihydrofolic acid,tetrahydrofolic acid, 5-methyltetrahydrofolic acid,5,10-methylenetetrahydrofolic acid, 5,10-methenyltetrahydrofolic acid,5,10-formiminotetrahydrofolic acid, 5-formyltetrahydrofolic acid(leucovorin), 10-formyltetrahydrofolic acid, 10-methyltetrahydrofolicacid, pharmaceutically compatible salts thereof, or a combination of twoor more thereof.
 39. The method as defined in claim 38 wherein at leastone of folate or tetrahydrobiopterin or arginine or a derivative thereofis in the natural stereoisomeric form of folate or tetrahydrobiopterinor arginine or a derivative thereof.
 40. A method for enhancing theactivity of tetrahydrobiopterin (BH₄), comprising administering folicacid, a folate or a derivative thereof.
 41. The method as defined inclaim 40 wherein folic acid, a folate or a derivative thereof is folicacid (pteroylmonoglutamate), one or more of the folylpolyglutamates,compounds in which the pyrazine ring of the pterin moiety of folic acidor of the folylpolyglutamates is reduced to give dihydrofolates ortetrahydrofolates, or derivatives of all the preceding compounds inwhich the N-5 or N-10 positions carry one carbon units at various levelsof oxidation, or pharmaceutically compatible salt thereof, or acombination of two or more thereof.
 42. The method as defined in claim41 wherein folate or a derivative thereof is dihydrofolic acid,tetrahydrofolic acid, 5-methyltetrahydrofolic acid,5,10-methylenetetrahydrofolic acid, 5,10-methenyltetrahydrofolic acid,5,10-formiminotetrahydrofolic acid, 5-formyltetrahydrofolic acid(leucovorin), 10-formyltetrahydrofolic acid, 10-methyltetrahydrofolicacid, pharmaceutically compatible salts thereof, or a combination of twoor more thereof.
 43. The method as defined in claim 42 wherein at leastone of folate or tetrahydrobiopterin or a derivative thereof is in thenatural stereoisomeric form.
 44. A pharmaceutical combination comprisingat least (1) folic acid or a folate; and (2) tetrahydrobiopterin (BH₄)or derivatives thereof
 45. The pharmaceutical combination as defined inclaim 44 wherein folic acid or a folate and tetrahydrobiopterin (BH₄) orderivatives thereof are present in one dosage within the range fromabout 0.1 to about 200 mg.
 46. A pharmaceutical combination comprisingat least (1) folic acid or a folate; and (2) tetrahydrobiopterin (BH₄);and (3) arginine. or derivatives thereof
 47. The pharmaceuticalcombination as defined in claim 46 wherein folic acid, folate or aderivative thereof and tetrahydrobiopterin (BH₄) and arginine arepresent in one dosage within the range from about 0.1 to about 200 mg.48. The pharmaceutical combination as defined in claim 46 wherein atleast one of folate or tetrahydrobiopterin or arginine or a derivativethereof is in the natural stereoisomeric form.